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Mayflies (Ephemeroptera) are among the oldest pterygote insects, with the earliest fossils dating back to the Late Carboniferous. Within mayflies, Leptophlebiidae are a highly diverse and widespread group, with approximately 140 genera and 640 species. Whereas taxonomy, systematics, and phylogeny of extant Leptophlebiidae are in the focus of extensive studies, little is known about leptophlebiid fossil taxa. Because fossil remains of Ephemeroptera in sedimentary rocks are relatively rare, inclusions of mayflies in amber are a unique source of information on their evolution and diversity in the past. Leptophlebiidae found in Cenozoic resins mostly belong to the subfamilies Leptophlebiinae (in Eocene Baltic amber) and Atalophlebiinae (in Miocene Dominican and Mexican ambers). In the present contribution, we confirm the first finding of the genus Calliarcys from Eocene Baltic amber by using Micro-CT, which allowed confirming its generic placement by visualizing diagnostic key characters otherwise hidden by a cloud of turbidity. The description and morphological reconstruction of the new fossil species Calliarcys antiquus sp. nov. and new molecular data of the extant Calliarcys humilis Eaton, 1881 from the Iberian Peninsula do not support a distinct subfamily Calliarcyinae, but reveal close relationships to the genera Leptophlebia and Paraleptophlebia, thus suggesting a phylogenetic position of Calliarcys within Leptophlebiinae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A recent electrical penetration graph (EPG) study identified a unique intracellular puncture that is associated with inoculation of the semipersistently transmitted, phloem-limited Beet yellows virus (BYV, Closterovirus) by the aphid Myzus persicae. This new aphid EPG pattern (named phloem-pd) always occurs shortly before phloem sieve element phase (PSEP) and has a similar voltage drop as PSEP, both of which are less than the voltage drop of standard-pds. Structure of pd subphase II-2 differs between phloem-pds and standard-pds. The objective of this study was to determine the type of phloem cell penetrated during phloem-pds. Stylets of M. persicae feeding on sugar beet (Beta vulgaris) were fixed in situ by cryofixation during phloem-pds, standard-pds and PSEP waveform E1. The cell penetrated by the stylet tips was then identified by confocal laser-scanning microscopy and micro-computed tomography. Sieve elements (SEs) or companion cells (CCs) were penetrated during phloem-pds, whereas cells other than SEs or CCs (mesophyll, bundle sheath cells and possibly phloem parenchyma) were penetrated during standard-pds. SEs were penetrated during waveform E1. The implications of these findings for inoculation of other phloem-limited viruses that currently are thought to be inoculated primarily during waveform E1 are discussed.

The Asian citrus psyllid (ACP), Diaphorina citri, is a harmful pest of citrus trees that transmits Candidatus Liberibacter spp. which causes Huanglongbing (HLB) (citrus greening disease); this is considered to be the most serious bacterial disease of citrus plants. Here we detail an anatomical study of the external and internal anatomy (excluding the reproductive system) using micro-computed tomography (micro-CT). This is the frst complete 3D micro-CT reconstruction of the anatomy of a psylloid insect and includes a 3D reconstruction of an adult feeding on a citrus leaf that can be used on mobile devices. Detailed rendered images and videos support frst descriptions of coxal and scapus antennal glands and sexual diferences in the internal anatomy (hindgut rectum, mesothoracic ganglion and brain). This represents a signifcant advance in our knowledge of ACP anatomy, and of psyllids in general. Together the images, videos and 3D model constitute a unique anatomical atlas and are useful tools for future research and as teaching aids., Kansas State University (KSU), University of Granada

Huanglongbing (HLB) (citrus greening disease) is one of the most serious bacterial diseases of citrus. It is caused by (1) Candidatus Liberibacter africanus, transmitted by Trioza erytreae and (2) C.L. asiaticus and C.L. americanus, transmitted by Diaphorina citri. As part of a multidisciplinary project on D. citri (www.citrusgreening.org), we made a detailed study, using micro-computed tomography, of the female abdominal terminalia, reproductive system (ovaries, accessory glands, spermatheca, colleterial (= cement) gland, connecting ducts, and ovipositor) and bacteriome, which we present here. New terms and structures are introduced and described, particularly concerning the spermatheca, ovipositor and bacteriome. The quality of images and bacteriome reconstructions are comparable, or clearer, than those previously published using a synchrotron or fuorescence in situ hybridisation (FISH). This study: reviews knowledge of the female reproductive system and bacteriome organ in D. citri; represents the frst detailed morphological study of D. citri to use micro-CT; and extensively revises existing morphological information relevant to psylloids, hemipterans and insects in general. High quality images and supplementary videos represent a signifcant advance in knowledge of psylloid anatomy and are useful tools for future research and as educational aids., Kansas State University (KSU) S15192.01, University of Granada, USDA-NIFA S15192.01 2014-70016-23028

The coffee berry borer (Hypothenemus hampei) is the most economically important insect pest of coffee globally. Micro-computed tomography (micro-CT) was used to reconstruct the respiratory system of this species for the first time; this is the smallest insect (ca. 2 mm long) for which this has been done to date. Anatomical details of the spiracles and tracheal tubes are described, images presented, and new terms introduced. The total volume and the relationship between tracheal lumen diameter, length and volume are also presented. The total length of the tracheal tubes are seventy times the length of the entire animal. Videos and a 3D model for use with mobile devices are included as supplementary information; these could be useful for future research and for teaching insect anatomy to students and the public in general., This paper benefitted from the sub-award agreement S15192.01 between Kansas State University (KSU) and the University of Granada, as part of a USDANIFA Award 2014-70016-23028 to S.J. Brown (KSU), “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease” (2015–2020).

15 pages, 14 figures, 2 tables, 1 appendix, We studied the anatomy and cytology of the midgut gland (MGl) of the common spider crab Maja brachydactyla Balss, 1922 at several life stages (zoea, megalopa, first juvenile, and adult) using dissection, histology, electron microscopy, computed tomography, and micro-computed tomography (micro-CT). In newly hatched larvae, 14 blind-end tubules form the MGl. The length of the tubules increases during the larval development. In the late megalopa, the number of tubules also increases. In adults, 35,000 to 60,000 blind-ending tubules comprise the MGl. In all life stages, a square-net network of muscle fibers surround the tubules. We describe five cell types in the MGl in all larval stages, which have a similar location, histology, and ultrastructure in larvae and adults: embryonary (E-) cells, resorptive (R-) cells, fibrillar (F-) cells, blister-like (B-) cells, and midget (M-) cells. Major difference between larval and adult cells is the larger size of the adult cells. Microapocrine secretion occurs from the microvilli of the B-cells. No ultrastructural changes were observed during larval development, which suggests that the function of each cell type might be similar in all life stages. The role of each epithelial cell type in larvae and adults is discussed, Financial support was provided by the Spanish Ministry of Economy and Competitiveness through the INIA project [grant number RTA 2011-00004-00-00] to G.G and a predoctoral fellowship to D.C. [FPI-INIA].

The coffee berry borer is the most devastating insect pest of coffee throughout the world. The insect spends most of its life cycle inside the coffee berry, which makes it quite difficult to observe its behaviour. Micro-computed tomography (micro-CT) was used to observe all developmental stages of the coffee berry borer inside coffee berries (Coffea canephora). An interesting oviposition pattern involving a sequential placement of eggs starting in the periphery of the seed and moving inwards was observed. Micro-CT should be useful in elucidating unknown life history aspects of other seed-feeding bark beetles as well as of bark and ambrosia beetles in general.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rivers in Mediterranean high mountains are especially vulnerable to climate change because these areas are characterized by extreme climatic conditions of snowy winters and relatively frequent summer droughts. Climate induced alterations in temperature and the magnitude of high and low river flows are expected to have significant effects on aquatic fauna. Here, we analysed changes in the caddisfly communities of the Sierra Nevada during a 20-year period on an altitudinal gradient range of 952–3050 m. Furthermore, we related these changes to an observed increase in air temperature and decrease in river flow over the last 40 years. Overall, caddisfly species richness increased but patterns varied depending on altitude in a non-linear shape. Richness increased in altitude with maximum values at sites of intermediate-high altitude (1800–2000 m). The effects of the observed climate change may be explained by the colonization of headwaters and middle reaches from mid-lowland species or by those from streams and rivers in nearby mountain chains at lower altitude. The observed richness increase and its association with environmental conditions suggest that mountains with a considerable altitudinal gradient may function as refuges for species and populations during periods of climatic change, which strength the importance of the conservation of mountainous habitat.

We carried out an in vivo study to evaluate the potential usefulness of a novel bioengineered bone substitute for the repair of palate defects in laboratory rabbits, using tissue-engineering methods. Our results showed that the use of a bioengineered bone substitute was associated with more symmetrical palate growth as compared to the controls, and the length and height of the palate were very similar on both sides of the palate, with differences from negative controls 4 months after artificial bone grafting for bone length. The histological analysis revealed that the regenerated bone was well organized and expressed osteocalcin. In contrast, bone corresponding to control animals without tissue grafting was immature, with areas of osteoid tissue and remodelling, as determined by MMP-14 expression. These results suggest that bone substitutes may be a useful strategy to induce the formation of a well-structured palate bone, which could prevent the growth alterations found in cleft palate patients. This opens a door to a future clinical application of these bone substitutes. Copyright © 2015 John Wiley & Sons, Ltd.

In Hemiptera, presumptive stylets for each consecutive postembryonic instar are manufactured prior to ecdysis to replace the ecdysial stylets discarded with the exuviae. With the discovery that the bacterium “Candidatus” Liberibacter solanacearum accesses the tissues involved in the stylet replacement process of the potato psyllid, a hypothesis was formed that the bacterium could adhere to the stylets of freshly emerged instars and hence gain access to the host plant when feeding is resumed. Although unproven, it was imperative that a model for stylet replacement be built. Stylet morphology and the stylet replacement process of the Asian citrus psyllid (ACP), vector of “C.” L. asiaticus, causal pathogen of citrus greening disease, are comparable to the potato psyllid model system. Morphology consists of a basal terminus with its tab-shaped auricle, a base, shaft, and an apical terminus. Each of the four auricles act as a platform for the replacement apparatus, which is compacted into a tight aggregate of cells, the ‘endcap’. As modeled, on apolysis of larval instar hypodermis, the aggregate ‘deconstructs’ and expands into a snail shell-shaped tube, the ‘atrium’, that houses the presumptive stylet as it is synthesized. Completed stylets then despool from the atrium and are fitted into their functional positions as the next instar emerges from its exuviae., Funding was provided by a grant from USDA-NIFA Award 2014- 70016-23028, 2015-2020, “Developing an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Disease”.

11 pages, 6 figures, supplementary material https://doi.org/10.1038/s41598-018-32709-3, Traditionally, the internal morphology of crustacean larvae has been studied using destructive techniques such as dissection and microscopy. The present study combines advances in micro-computed tomography (micro-CT) and histology to study the internal morphology of decapod larvae, using the common spider crab (Maja brachydactyla Balss, 1922) as a model and resolving the individual limitations of these techniques. The synergy of micro-CT and histology allows the organs to be easily identified, revealing simultaneously the gross morphology (shape, size, and location) and histological organization (tissue arrangement and cell identification). Micro-CT shows mainly the exoskeleton, musculature, digestive and nervous systems, and secondarily the circulatory and respiratory systems, while histology distinguishes several cell types and confirms the organ identity. Micro-CT resolves a discrepancy in the literature regarding the nervous system of crab larvae. The major changes occur in the metamorphosis to the megalopa stage, specifically the formation of the gastric mill, the shortening of the abdominal nerve cord, the curving of the abdomen beneath the cephalothorax, and the development of functional pereiopods, pleopods, and lamellate gills. The combination of micro-CT and histology provides better results than either one alone, Financial support was provided by the Spanish Ministry of Economy and Competitiveness through the INIA project (grant number RTA2011-00004-00-00) to G.G. and a pre-doctoral fellowship to D.C. (FPI-INIA)

Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels. This article is protected by copyright. All rights reserved.

Several studies on global change over the next century predict increases in mean air temperatures of between 1°C to 5°C that would affect not only water temperature but also river flow. Climate is the predominant environmental driver of thermal and flow regimes of freshwater ecosystems, determining survival, growth, metabolism, phenology and behaviour as well as biotic interactions of aquatic fauna. Thus, these changes would also have consequences for species phenology, their distribution range, and the composition and dynamics of communities. These effects are expected to be especially severe in the Mediterranean basin due its particular climate conditions, seriously threatening Southern European ecosystems. In addition, species with restricted distributions and narrow ecological requirements, such as those living in the headwaters of rivers, will be severely affected. The study area corresponds to the Spanish Mediterranean and Balearic Islands, delimited by the Köppen climate boundary. With the application of the MEDPACS (MEDiterranean Prediction And Classification System) predictive approach, the macroinvertebrate community was predicted for current conditions and compared with three posible scenarios of watertemperature increase and its associated water flow reductions. The results indicate that the aquatic macroinvertebrate communities will undergo a drastic impact, with reductions in taxa richness for each scenario in relation to simulated current conditions, accompanied by changes in the taxa distribution pattern. Accordingly, the distribution area of most of the taxa (65.96%) inhabiting the mid-high elevations would contract and rise in altitude. Thus, families containing a great number of generalist species will move upstream to colonize new zones with lower water temperatures. By contrast, more vulnerable taxa will undergo reductions in their distribution area., This work was funded by GUADALMED-II (REN2001-3438-C07-06/HID), a project of excellence from “Junta de Andalucía” (RNM-02654/FEDER), the Spanish “Ministerio de Ciencia e Innovación” (CGL2007-61856/BOS), projects and a collaboration agreement between the “Spanish Ministerio de Medio Ambiente, Medio Rural y Marino” and the University of Granada (21.812-0062/8511).

Climate change will cause relict alpine glaciers to disappear within decades. Associated high-altitude streams will face significant hydrological changes that might affect population genetic diversity of lotic species. In a recent study of glacier-fed streams in the Pyrenees, Finn et al. (2013) predicted that a large proportion of regional genetic diversity of the mayfly Baetis alpinus would be lost as glacial meltwater sources disappear. We expanded the analysis of Finn et al. to include genetic data (mitochondrial barcoding region) collected from B. alpinus occupying recently deglaciated locations including multiple basins in 2 mountain ranges (Sierra Nevada and Picos de Europa) on the Iberian Peninsula and 1 additional, deglaciated Pyrenean basin. We hypothesized that regional genetic diversity at scales of entire mountain ranges (γ diversity) and population structure within ranges (β diversity) would be lower in recently deglaciated mountain ranges than in the still-glaciated Pyrenees. For 4 c...

A new Italian earthworm morphologically close to the similarly large and anecic Eophila tellinii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750 km2 from East to West on the Asiago Plateau and Vittorio Veneto Hills, from North to South on mounts Belluno Prealps (Praderadego and Cesen), Asiago, Grappa and onto the Montello foothills. This range abuts that of Eophila tellinii in northern Friuli Venezia Giulia region. Known localities of both E. tellinii and E. crodabepis sp. nov. are mapped. mtDNA barcoding definitively separates the new western species from classical Eophila tellinii (Rosa, 1888).

The nymphal biology of a population of Torleya major (Klapalek) in southern Iberian Peninsula was studied. An atypical life cycle pattern is described, with eggs hatching in August producing a fast-developing cohort with adults emerging in autumn and a second slow-developing cohort with adults emerging in spring of the following year. Nymphal growth occurred primarily in summer–autumn (in the first cohort) and in spring (in the second). The origin of such a life history is discussed. Nymphs were collector-gatherers, consuming mainly detritus. Although ontogenetic shifts on the use of trophic resources were detected, similar food was utilized during the months when both cohorts cohabited, eliminating the possibility that the rapid growth of the first cohort could be related to the utilization of different food resources.

The Asian citrus psyllid (ACP), Diaphorina citri, is a major vector of the bacteria Candidatus Liberibacter asiaticus and C.L. americanus, which cause Huanglongbing disease (HLB) (aka Citrus greening disease), considered the most serious bacterial disease of citrus trees. As part of a multidisciplinary project on psyllid biology (www.citrusgreening.org), the results presented here concern a detailed anatomical study of the male reproductive system (testes, seminal vesicles, accessory glands, sperm pump, connecting ducts, and aedeagus) using micro-computed tomography (micro-CT). The study summarizes current knowledge on psyllids male reproductive system and represents significant advances in the knowledge of ACP anatomy., This work was supported by USDA-NIFA Award 2014-70016-23028 ªDeveloping an Infrastructure and Product Test Pipeline to Deliver Novel Therapies for Citrus Greening Diseaseº, 2015-2020

We studied aspects of the nymphal biology and ecology of 3 Perlodidae species (Guadalgenus franzi, Hemimelaena flaviventris, and Isoperla curtata) in a Mediterranean seasonal stream in the southern Iberian Peninsula. Their life-history strategies were greatly influenced by the characteristics of their environment, i.e., a summer dry period with relatively warm temperatures, but strategies differed among species. Guadalgenus franzi was semivoltine and probably underwent nymphal quiescence when the stream was dry. Hemimelaena flaviventris and I. curtata had relatively short univoltine life cycles that overlapped, but I. curtata was slightly ahead of H. flaviventris. Both species passed the dry period in the egg stage, probably with a diapause phase. Growth rates of H. flaviventris and I. curtata peaked before emergence, whereas growth rate of G. franzi peaked immediately before and after the dry period. The 3 species had relatively short flight periods compared with other species from seasonal streams. Guadalgenus franzi and I. curtata were mainly scrapers that fed on diatoms, whereas H. flaviventris was mainly predatory. Diet changed somewhat in relation to size, and prey electivity patterns differed among species. All 3 species preferred Chironomidae, but G. franzi preferred Chironomidae principally when smaller. All 3 species had high secondary production relative to other stonefly species from both temporary and permanent waters.

We studied some ecological aspects of two taxa of Nemouroidean stonefl ies (Tyrrhenoleuctra sp. and Brachyptera vera cordubensis) from a seasonal stream in Southern Europe. Tyrrhenoleuctra sp. shows a univoltine life cycle with a nymphal dormancy during the drought, and a long fl ight period. The eggs of this species are fully developed when laid, and hatch rapidly after laying, indicating a certain degree of ovoviviparism. The fi rst instar nymph is described for the fi rst time. In contrast, B. vera cordubensis shows a fast seasonal univoltine life cycle with an egg dormancy. The feeding behaviour study reveals that Tyrrhenoleuctra sp. can be catalogued mainly as a gatherer-collector while B. vera cordubensis can be classifi ed mainly as a scraper, although neither of them belong solely to a single functional group. No signifi cant changes are detected in the diet in relation to nymphal size, although larger individuals of B. vera cordubensis ingest more CPOM than small ones. Both taxa had similar biomass, but B. vera cordubensis had a higher secondary production and annual P/B rate.

Burrowing is a common behavioural adaptation of lotic freshwater invertebrates to avoid the effects of current. This behaviour is accompanied by morphological adaptations. This also applies to the larvae of the stonefly Leuctra geniculata (Stephens, 1836) and the mayfly Ephemera danica Muller, 1764, both colonising habitats within the substrate and adapted to burrow in it. We have studied their life cycles and their relation with water temperature and day-degrees, feeding and secondary production. The stonefly had a univoltine life cycle with a larval development of 8 months and with an egg incubation period longer than previously reported. The possibility of an egg diapause stage is discussed. The mayfly was semivoltine, completing its larval development in 22 months. Both species mainly fed on detritus, but also ingested a high quantity of CPOM and some other minor components. Annual secondary production in both species was relatively high, being higher in the stonefly.

In mid latitudes, such as the Mediterranean region, one consequence of global climate change will be a progressive shift of permanent to temporary water courses, altering the life histories of present species or making them disappear. Presented here is a study on the life history of a mayfly species, Serratella ignita, in two Mediterranean streams, one permanent and one temporary. The research focuses on life history parameters such as life cycle, nymphal growth pattern, feeding type, biomass and secondary production. The life cycle was univoltine, showing a fast seasonal nymphal growth during four months in the permanent stream and five months in the temporary stream, with a long egg stage with diapause. The cumulated day-degrees during nymphal growth were similar between streams. Both populations behaved mainly as gatherer-collectors, but the population from the temporary stream also played an important role as a scraper. Animal matter was also consumed by nymphs from both populations. Gut content analysis showed that both S. ignita populations were opportunist/generalists, where changes in diet were correlated with size. Biomass and secondary production were notably higher in the permanent stream, but they were also high in the temporary stream. These data show that S. ignita had a high fitness in both streams. Our results support the idea that species such as this one may cope with future global climate change by maintaining, or even increasing, their populations at the expense of more stenoecic species.

Aim To examine how the employment of both community- and population-level approaches can provide a wider view of the importance of contemporary and historical factors on current species distribution. We posit that community ecology should provide more information about contemporary factors, whereas population genetics should provide better information about historical factors. Location Rivers of the western Mediterranean Basin, including four subregions differing in geological history: the Iberian Plate, Transitional, Betic and Rif. Methods For a community-level approach, Trichoptera richness and community composition were compared between subregions using species accumulation curves and a correspondence analysis. For a population-level approach, the mtDNA cytochrome C oxidase subunit I (COI) gene of specimens of the Trichoptera midstream-lowland species Chimarra marginata (L.) was sequenced and analysed using phylogeographical methods. Results The community approach revealed that historical events had more influence on headwater communities than contemporary ecological factors, whereas historical events had negligible influence on midstream-lowland communities. In midstream-lowland sites, however, the population approach showed that the genetic structure of C. marginata differed significantly between subregions and revealed patterns of historical gene migration. In terms of species richness, the Rif subregion had the lowest value per basin due to local climatic features and isolation. Main conclusions Both community- and population-level approaches yielded information about the effects of historical factors on species distribution. However, the importance of historical events on current Trichoptera communities depends on the river zonation. Unlike headwater sites, midstream-lowland sites showed signs of historical events at the population level but not at the community level at the scale used, indicating that both approaches should be employed together in biogeographical studies. Lack of detection of historical events at the community level does not necessarily mean that they are negligible. Most likely, the organizational level used is not appropriate. We also stress the importance of implementing conservation measures for rivers in the western Mediterranean, especially under future scenarios of climate change and human disturbances in the Mediterranean Basin.

In Spain, a national project known as GUADALMED, focusing on Mediterranean streams, has been carried out from 1998 to 2005 to implement the European water framework directive (WFD) requirements. One of the main objectives of the second phase of the project (2002-2005) was to develop a predictive system for the Spanish Mediterranean aquatic macroinvertebrate communities. A combined-season (spring, summer, and autumn) predictive model was developed by using the latest improvements on the selection of best predictor variables. Overall model performance measures were used to select the best discriminant function (DF) models, and also to evaluate their biases and precision. The final predictive model was based on the best five DF models. Each one of these models involved eight environmental variables. Final observed (O), expected (E), and O/E values for the number of macroinvertebrate families (NFAM) and two biotic indices (IBMWP and IASPT) were calculated by averaging their values, previously weighted by the quality of each DF model. Regression analyses among the final O and E values for the calibration dataset showed a high proximity to the ideal theoretical model, where the final E values explained 73-84% of the variation present in the macroinvertebrate communities of the Spanish Mediterranean watercourses. The ANOVA performed among the reference (calibration and validation) and test datasets showed clear differences for the O/E values. Finally, the assessments carried out by the predictive model were sensitive to anthropogenic pressure present in the study area and allowed the definition of five ecological status classes according to the WFD requirements.

We monitored two high mountain streams from the Sierra Nevada (Southern Spain) monthly throughout an annual cycle, measuring and analyzing physical and chemical parameters. Temperature was hourly registered. We studied life cycles and larval feeding of four mayfly species [Serratella ignita (Poda, 1761), Serratella spinosa nevadensis (Alba-Tercedor, 1981), Alainites muticus (Linnaeus, 1758), and Baetis alpinus Pictet, 1843] and two stonefly species [Amphinemura triangularis (Ris, 1902) and Capnioneura mitis Despax, 1932]. Some species changed their strategies (growth rate, life cycle duration, and/or voltinism) in accordance with different conditions (depending on the altitude and temperature). The gut content data showed that species fed mainly on detritus (as collector-gatherers or as shredders), except B. alpinus that behaved as a scraper in one of the streams. Differences in the larval diet of the same species in different streams, and even through its life, support the idea of not using general models to extrapolate the function of a species from a higher taxonomical level.

Summary 1. According to the guidelines of the European Water Framework Directive, assessment of the ecological quality of streams and rivers should be based on ecotype-specific reference conditions. Here, we assess two approaches for establishing a typology for Mediterranean streams: a top-down approach using environmental variables and bottom-up approach using macroinvertebrate assemblages. 2. Classification of 162 sites using environmental variables resulted in five ecotypes: (i) temporary streams; (ii) evaporite calcareous streams at medium altitude; (iii) siliceous headwater streams at high altitude; (iv) calcareous headwater streams at medium to high altitude and (v) large watercourses. 3. Macroinvertebrate communities of minimally disturbed sites (n = 105), grouped using UPGMA (unweighted pair-group method using arithmetic averages) on Bray–Curtis similarities, were used to validate four of the five ecotypes obtained using environmental variables; ecotype 5, large watercourses, was not included as this group had no reference sites. 4. Analysis of similarities (anosim) showed that macroinvertebrate assemblage composition differed among three of the four ecotypes, resulting in differences between the bottom-up and top-down classification approaches. Siliceous streams were clearly different from the other three ecotypes, evaporite and calcareous ecotypes did not show large differences in macroinvertebrate assemblages and temporary streams formed a very heterogeneous group because of large variability in salinity and hydrology. 5. This study showed that stream classification schemes based on environmental variables need to be validated using biological variables. Furthermore, our findings indicate that special attention should be given to the classification of temporary streams.

We carried out an in vivo study to evaluate the potential usefulness of a novel bioengineered bone substitute for the repair of palate defects in laboratory rabbits, using tissue-engineering methods. Our results showed that the use of a bioengineered bone substitute was associated with more symmetrical palate growth as compared to the controls, and the length and height of the palate were very similar on both sides of the palate, with differences from negative controls 4 months after artificial bone grafting for bone length. The histological analysis revealed that the regenerated bone was well organized and expressed osteocalcin. In contrast, bone corresponding to control animals without tissue grafting was immature, with areas of osteoid tissue and remodelling, as determined by MMP-14 expression. These results suggest that bone substitutes may be a useful strategy to induce the formation of a well-structured palate bone, which could prevent the growth alterations found in cleft palate patients. This opens a door to a future clinical application of these bone substitutes. Copyright © 2015 John WileySons, Ltd.

The southeastern mountains of Spain represent the southernmost limit of the genus Austropotamobius and the species A. pallipes (Lereboullet). The taxonomic position of this isolated crayfish in southern Spain is not clear, being genetically close to A. italicus, but morphologically distinct. A severe decline occurred during the 1980s, especially due to expansion of the alien species Procambarus clarkii, a North American freshwater crayfish and a vector of the aphanomycosis disease. In order to design a strategy for native crayfish conservation, recent trends in native crayfish populations, influence of isolation and habitat variables on their survival and possibilities for their recovery through restocking were studied. A decline in populations was observed between 1991 and 2002 (from 26 to 14 populations), and a total extinction could be predicted within the next 13 years. Two well-defined periods of rapid extinction rates were detected, 1991–1994 (1.25 populations/year) and 1999–2002 (1.50 populations/year). Main causes of extinction for these two periods were invasion by P. clarkii and mortality by unknown causes, respectively. The isolation variables had some positive effects on survival of populations but these cannot offer a sufficient guarantee, since several cases of extinction can be affected by a large component of stochastic factors, including random catastrophes. On the other hand, survival trials and restocking experiments showed that it was possible to recover lost habitats, when P. clarkii was absent and environmental conditions were good. It is concluded that it is possible to avoid extinction of the native crayfish populations; however, the conservation strategies must be based on an urgent stocking/restocking program.

As a result of studies of material of Ecdyonurus from Spain, nymphs and adults of two new species, E. baeticus sp. n. and E. olgae sp. n., are described. Three species described by Navas must be considered as nomina dubia (E. diversus Navas, 1923, E. excelsus Navas, 1927 and E. fluminum var. speciosus Navas, 1915). Variability in adults of E. venosus is figured and discussed.

After an intensive study, we concluded that the species described by Alba-Tercedor and Sanchez-Ortega (1982) from the foothills of the Sierra Nevada mountains in Southern Spain as Torleya cf. belgica, actually belongs to a new species which we name Torleya nazarita sp. n. The new species is closely related to Torleya major (Klapalek, 1905), which up to now was considered to be the only European representative of the genus. Description and figures of the nymph and imago and the features distinguishing the new species from other species of the genus are given.

A new species of Hydropsyche of the instabilis-group, H. fontinalis sp. n., from the southern Iberian Peninsula is described. Diagnostic features of the male, female and larva (5th instar) are described and illustrated, and additional information on larval habitat and ecological notes are included.

The former and present distribution of white clawed crayfish (Austropotamobius pallipes) in the province of Granada (southern Spain) is studied. Before 1980 it was widely distributed but at present only 16 populations exist. The decline is related to the presence of the freshwater red-swamp crayfish (Procambarus clarkii), an American species, vector of the aphanomycosis disease, introduced to the Iberian Peninsula in 1974 and now widely distributed in the watercourses and marshes of southern Spain. To establish an appropriate conservation policy for A. pallipes at its southernmost distribution limit, we studied watercourses from two river basins, Genil and Guadiana Menor, (tributaries of the Guadalquivir River). P. clarkii inhabits the medium to lower reaches of these two river basins (with its upper limit at 820 m a.s.l.). The distribution of this species was best explained by the effect of three of the 12 analyzed variables: altitude, water-current and minimum winter temperatures. From our results, the repopulation of the native crayfish is almost impossible in those reaches inhabited by P. clarkii. However, based on the habitat selection study, it is clear that upper reaches are unsuitable for the red-swamp crayfish, where the native white-clawed crayfish may have greater survival possibilities, and these sites can be used for future restocking projects.